Nitro substituted chemiluminescent labels and their conjugates, and assays therefrom

ABSTRACT

A novel chemiluminescent labeling compositions comprising an ester, thiolester or amide covalently and jointly bonded to (1) a carbon of a heterocyclic ring or ring system that is susceptible to attack by peroxide or molecular oxygen and (2) an aryl ring or ring system wherein the heterocyclic ring or ring system is distinguished by a heteroatom thereof in an oxidation state which causes the attacked carbon atom to form an intermediate that decays and produces chemiluminescence; the aryl ring or ring system contains at least three substituents on a six-member aromatic hydrocarbon that together sterically and electronically hinder hydrolysis of the linkage, which substituents involve ortho substituent groups on the aryl in conjunction with --NO 2  meta or para substituents thereon. Included are the chemiluminescent labeling composition conjugated with a specific binding material; a chemiluminescent assay comprising the conjugate; and a chemiluminescent assay kit comprising the conjugate with the capability of conducting the assay.

RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.140,040, filed Dec. 31, 1987, now abandoned, and, copending applicationSer. No. 291,843, filed Dec. 29, 1988, now abandoned and copendingapplication Ser. No. 418,956, filed Oct. 10, 1989, now abandoned.

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to unique chemiluminescent labeling compounds,conjugates containing associated versions of the labeling compounds,assays and kits for performing such assay utilizing the conjugates. Thelabeling compounds contain special sterically-hindered aryl heterocyclicesters, thiolesters and amides by virtue of the presence of a nitrosubstituent directly attached to the aryl group.

BACKGROUND TO THE INVENTION

The literature describes classes of compounds that give off light or"luminesce" by reaction through chemical treatment. The compounds thathave this capability are termed chemiluminescent materials. Theirdissociation is typically caused by treatment with peroxide or molecularoxygen at high pH. Light is produced by the decay of the transient("intermediate") structure formed by peroxide or molecular oxygenreaction at an sp² or sp³ hybridized carbon in the compound that is partof a chain or a ring or ring system.

As the literature indicates, any series of reactions which produce theintermediate: ##STR1## will lead to moderate to strongchemiluminescence. F is a structure such that the product carbonylderivative ##STR2## is fluorescent and X is a good leaving group,usually with XH, for efficient chemiluminescence, having a pK_(a) ofabout >11, preferably <11, and most preferably, from about 5 to about 8.The reaction may require base catalysis.

The intermediate can be prepared (in isolable or transient form,depending on F) from species such as: ##STR3## See Endeavour, 23, No.117 (1973) p. 140, The Chemistry of Bioluminescence in "Bioluminescencein Action" (P. J. Herring, ed.), Academic Press, London, 1978 (pp.64-5), Proc. R. Soc. Lond., B 215, p. 256 (1982), Progress in OrganicChemistry, (W. Carruthers and J. K. Sutherland, eds.), Butterworth,London (1973), p. 261, all authored by F. McCapra.

For example, chemiluminescent aryl esters that contain such hybridizedcarbon, termed a labeling compound, react according to the followinggeneral reaction: ##STR4## where A is an aryl ring or ring system and Bis a heterocyclic ring or ring system. In this reaction, --O--A, theleaving group, is cleaved by perhydrolysis resulting in steps leading tothe transient intermediate, B=O, that proceeds to decay generatingluminescence.

The characteristics of some of these chemiluminescent compounds, theirchemistry of manufacture, and other factors relating to them, are morefully described by McCapra, "Chemiluminescence of Organic Compounds," inProgress in Organic Chemistry, vol. 8, Carruthers and Sutherland ed.,Wiley & Sons (1973); Kohen, Bayer, Wilechek, Barnard, Kim, Colleins,Beheshti, Richardson and McCapra, "Development Of Luminescence-BasedImmunoassays For Haptens And For Peptide Hormones," pp. 149-158, inAnalytical Applications Of Bioluminescence and Chemiluminescence,Academic Press, Inc. (1984); Richardson, Kim, Barnard, Collins andMcCapra, Clinical Chemistry, vol. 31, no. 10, pp. 1664-1668 (1985);McCapra, "The Application of Chemiluminescence in Diagnostics," 40thConference of the American Association of Clinical Chemists, NewOrleans, La., Jul. 28, 1988; McCapra, "The Chemiluminescence Of OrganicCompounds," Quarterly Reviews, vol. 20, pp. 485-510 (1966); McCapra,"The Chemiluminescence Of Organic Compounds," Pure and AppliedChemistry, vol. 24, pp. 611-629 (1970); McCapra, "The chemistry ofbioluminescence," Proceedings Of Royal Society, vol. B215, pp. 247-278(1982); McCapra and Beheshti, "Selected Chemical Reactions That ProduceLight," Bioluminescence and Chemiluminescence: Instruments andApplications, CRC Press, vol. 1, Chapter 2, pp. 9-37 (1985); McCapra,"Chemiluminescent Reactions of Acridines," Chapt. IX, Acridines, R. M.Acheson, Ed., pp. 615-630, John Wiley & Sons, Inc. (1973); McCapra,"Chemical Mechanisms in Bioluminescence," Accounts Of Chemical Research,vol. 9, no. 6, pp. 201-208 (June 1976); and in many other publicationsand presentations on the subject.

As noted in the above literature, chemiluminescent compounds of avariety of structures have been projected as labels for a variety ofassays including immunoassays (in this respect, see U.S. Pat. Nos.4,383,031, 4,380,580 and 4,226,993). The esters, thiolesters and amides,alone or conjugated (i.e., chemically coupled to another material), areespecially desirable forms of chemiluminescent labels. However, theylose their luminescence capability over time in an aqueous systembecause they hydrolyze to products that are not available to the assay.Until recently, these compounds have not been used in commercial assays.Until this invention, the ester, thiolester and amide forms of thisclass of materials lacked sufficient hydrolytic stability to be storedin the most convenient form over an extended period of time, which is asa component of an aqueous system.

It is well understood in chemistry that carboxylic acid esters,thiolesters and amides are susceptable to hydrolytic attack resulting inthe formation of the carboxylic acid and the hydroxy, mercapto or aminocomponent that is the theoretical or actual precursor to the ester,thiolester or amide. Hydrolysis is more pronounced under acid or basicconditions. It is also recognized in chemistry that certain levels ofhydrolysis can be inhibited by the inclusion of properly positionedbulky groups that chemically sterically hinder those linkages, seeNishioka et al., J. Org. Chem., vol. 40, no. 17, pp. 2520-2525 (1975),Fujita et al., "The Analysis of the Ortho Effect," Progress in PhysicalOrganic Chemistry, 8, pp. 49-89 (1976), Morrison and Boyd, OrganicChemistry, 5th Ed., pp. 842-843 (1987) and March, Advanced OrganicChemistry, 3rd Ed., page 240 (1985). According to March:

"Another example of steric hindrance is found in 2,6-disubstitutedbenzoic acids, which are difficult to esterify no matter what theresonance or field effects of the groups in the 2 or the 6 position.Similarly, once the 2,6-disubstituted benzoic acids are esterified, theesters are difficult to hydrolyze." (Emphasis in the original)

The difficulty in esterification is not the same in making esters from2,6-substituted phenols, but the general principles described by Marchare applicable to enhancing the hydrolytic stability of the resultantester so long as the ortho substitutions are electron donating. As thisinvention demonstrates, effective levels of hydrolytic stability requirethe presence of a select level of electron withdrawing chemical effectin conjunction with (and in addition to) traditional chemical sterichindrance factors.

The functional electron withdrawing or electron donating characteristicsof a group in an organic compound is conventionally measured relative tohydrogen. This relative ranking accepts that all groups on a moleculewill provide some slectron withdrawing effect, and distinquishes them bythe nature of impact the group has on the molecule's performance. Anelectron withdrawing functional group, characterized by a positivenumber, will draw electrons to itself more than hydrogen would if itoccupied the same position in the molecule. The opposite occurs with an"electron donating group," a lesser electron withdrawing group whichchemical convention characterizes by a negative number. Sigma paravalues (σ_(p)) are the relative measurement of electron withdrawing orelectron donating qualities of a functional group in the para positionon benzoic acid. See March, Advanced Organic Chemistry, 3rd Edition,Publ. by John Wiley & Sons, New York, N.Y. (1985) at pp. 242-250 and617-8. Tables of σ_(p) values for various groups can be found in Hanschet al., J. Med. Chem. 16(11):1209-1213 (1973) and Hansch and Leo,"Substituent Constants for Correlation Analysis in Chemistry andBiology," Ch. 6, pp. 49-52 (John Wiley & Sons, New York 1979). The σ_(p)values reported in the Hansch articles are relied on herein incharacterizing relative values for groups both in the meta and paraposition.

The function of chemiluminescent labels in assay applications involvesthe coupling of the label compound to a substrate molecule. Suchcoupling can be achieved by solvent interraction (e.g., molecularcompatibility), any heterolytic or homolytic mechanism induced bychemical means and influenced by physical effects, such as time,temperature and/or mass action. For example, the reaction can benucleophilic or electrophilic, or it can involve free radicalmechanisms. In the broadest perspective, the coupling can be viewed asachievable via strong to weak bonding forces.

A chemiluminescent label in assays is an associated moiety of a bindingmaterial. The moiety is derived from a chemical compound which, as such,possesses chemiluminescent capabilities. Hereinafter, the term moiety asapplied to the label as such, is a reference to the compound prior tobeing associated with a binding material. The term associated isintended to include all or any of the mechanisms for coupling the labelto the substrate molecule.

The term "functional" in chemistry typically refers to a group thatinfluences the performance of a chemical or constitutes the site forhomolytic or heterolytic reactions. For example, a functional alkylsubstituent, used in the context of interreactions through thatsubstituent, means an alkyl group substituted so that it can effect thatreaction. But an alkyl group termed functional for the electroniceffects it induces in the molecule is a reference to the alkyl group perse.

THE INVENTION

This invention relates to unique nitro containing chemiluminescentlabeling compounds, conjugates containing associated versions of thelabeling compounds, assays and kits for performing such assay utilizingthe conjugates. The labeling compounds contain specialsterically-hindered aryl heterocyclic substituted esters, thiolestersand amides that contain nitro groups directly bound to the aryl moiety.

This invention relates to novel chemiluminescent labeling compositionsand their conjugates with specific binding materials that are normallymaintained in an aqueous medium. These compositions and the conjugatesfind special application in specific binding assays because thechemiluminescent compound, i.e., the labeled moiety, has increased andunique stability in aqueous mediums and exceptional chemiluminescenceproperties.

The novel root compound of the invention is a chemiluminescent compoundcharacterized by the presence an aryl ester, thiolester or amide of acarboxylic acid substituted heterocyclic ring that is susceptible tochemical attack (such as by oxidic attack) to dissociate theheterocyclic ring to a transient compound. The heterocyclic ring is ringcarbon-bonded to the carbonyl of the ester, thiolester and amide moietyand possesses a heteroatom in an oxidation state that allowschemiluminescence by dissociating a compound at the carbon bonded to thecarbonyl ("intermediate") that decays to produce chemiluminescence. Thearyl ring or ring system is ring carbon-bonded to the oxygen or sulfurof the ester or thiolester, as the case may be, and contains at leastthree substituents on a six-member ring. The substitution on thesix-member ring comprises three or more groups acting in concert tosterically and electronically hinder hydrolysis of the ester, thiolesteror amide linkage. Significant to this invention is the presence ofdiortho electron donating substitution on the aryl unit in conjunctionwith a nitro meta and/or para substituent. It is this combination thatcauses the chemiluminescent label compound to have uniquely highhydrolytic stability.

Also in accordance with the present invention are conjugates of thelabeling composition, assay systems utilizing the conjugates, and assaykits incorporating such chemiluminescent labels.

In particular, this invention relates to a hydrolytically stableheterocyclic composition capable of chemiluminescent properties whenlabeled (i.e., affixed as a label) to a specific binding material bychemically-induced dissociation, comprising

(a) an aryl ring,

(b) a sterically-hindered ester, thiolester or amide linkage moiety withenhanced hydrolytic stability, and

(c) a heterocyclic organic ring moiety,

in which

(1) the carbonyl carbon of (b) is covalently bonded to a carbon atom (x)of (c) and the remaining free valence of (b) is carbon bonded to anaromatic ring carbon atom (y) of (a),

(2) (a) contains at least three substituent groups hindering hydrolysisof (b), two of which are electron donating and located on the ringcarbon atoms adjacent to (y), the remainder is nitro that is meta orpara to (y), and

(3) (c) contains

(i) at least one ring carbon atom adjacent to said ring carbon atom

(x), and

(ii) a ring member heteroatom in an oxidation state that provides suchchemiluminescence properties.

Also, this invention contemplates hydrolytically stable conjugatespossessing chemiluminescent properties by chemical dissociation,comprising a chemiluminescent label bonded to a specific bindingmaterial that contains

(a) an aryl ring,

(b) a sterically-hindered ester or thiolester linkage moiety withenhanced hydrolytic stability, and

(c) a heterocyclic organic ring moiety,

in which

(1) the carbonyl carbon of (b) is covalently bonded to a carbon atom (x)of (c) and the remaining free valence of (b) is carbon bonded to anaromatic ring carbon atom (y) of (a),

(2) (a) contains at least three substituent groups hindering hydrolysisof (b), two of which are electron donating and located on the ringcarbon atoms adjacent to (y), the remainder is nitro that is meta and/orpara to (y), and

(3) (c) contains

(i) at least one ring carbon atom adjacent to said ring carbon atom (x),and

(ii) a ring member heteroatom that is in an oxidation state wherebyreaction of molecular oxygen or a peroxide with said composition formsan intermediate which decays to produce chemiluminescence.

The invention encompasses a method for assaying the presence of ananalyte in a sample. The method comprises contacting an analyte with theaforementioned chemiluminescent-labeled specific binding material (the"conjugate"), inducing chemiluminescence by decay of an intermediatedissociated from the conjugate, and measuring luminescence therefrom toassay the analyte.

In keeping with the inventive chemiluminescent-label's function ofassaying, the invention embodies a specific binding assay kit comprisinga vial containing a conjugate possessing chemiluminescent properties bychemically induced dissociation and containing the aforementionedchemiluminescent label bonded to a specific binding material.

The invention recognizes that hydrolytic stability of a chemiluminescentlabel composition that utilizes aryl ester, thiolesters and amides, asdefined herein, linked to heterocyclic carboxy compounds, is affected bytwo factors. The first is the utilization of diortho substitution on thearyl ring of a kind that traditionally contributes to hydrolyticstability. This is the "bulky group" steric hindrance effect noted byMorrison and Boyd, supra. In the context of sigma values, these bulkygroups are typically classed as electron donating. The second is theutilization of meta and/or para substitution on the same ring thatuntraditionally contributes to hydrolytic stability. This lattersubstitution is a nitro group directly bonded to the ring. Thiscombination of steric hindrance and the nitro group provides materiallysuperior hydrolytic stability to the labeling composition than othersubstituents.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned above, proper positioning of bulky groups in an esterenhances the ester's hydrolytic stability. In the case of an aryl ester,steric hindrance is expected with bulky groups that block the estermoiety. Those groups may be provided on the aryl group in the orthopositions relative to the juncture with the ester moiety. Groups thatmay be dormant to any other reaction may not provide steric hindrancebecause of its chemical nature. For example, a group in a position alphato a carbonyl moiety of an ester group that is electron withdrawing(such as an alpha chloro group) could adversely affect the hydrolyticstability of the ester group. However, a methyl electron donating groupin the same position enhances hydrolytic stability. Thus, the relativeposition of the bulky group and its chemical nature is important tosteric hindrance. As a result, the typical bulky group provided in theortho position of an aryl ester for steric hindrance is electrondonating. An electron withdrawing group would be expected to adverselyaffect the hydrolytic stability of the aryl ester. The presence of anelectron withdrawing group substituted on an aryl ester would beexpected to, to some degree, as a result of the field effects theyintroduce, reduce the hydrolytic stability of the ester, even if it isotherwise substituted with electron donating groups in the orthopositions. The presence of the electron withdrawing group would beexpected to reduce the electron flow provided by the ortho electrondonating groups to the ester group.

It has been surprising found that the presence of nitro groups in arylesters in positions meta and/or para to the ester linkage contributesignificantly to the hydrolytic stability of the ester group. Thefollowing experiments demonstrates this point.

A small amount (about 0.1 mg) of a substituted phenyl ester ofacridinium carboxylate fluorosulfonate chemiluminescent label, depictedin the following table, was dissolved in 100 μl of N,N-dimethylformamide(DMF). This solution was serially diluted (in the v/v ratio of 1:100) ina pH 6.0 phosphate buffer containing 0.4% BSA (bovine serum albumin) and0.001% Thimerosal®, as preservative. Chemiluminescent counts between100,000 and about 400,000 were obtained. About 2 to 5 ml were incubatedat 37° C. in a clear 5 ml vial capped with a rubber stopper and wrappedwith Parafilm®. Three 20 μl samples of each label were counted in aBerthold® luminometer that was checked at various time intervals in thecourse of 14 days. The percent recovery of chemiluminescent counts after14 days is set forth in the following table, as well as the relativeratio against a σ_(p) value of 0:

    __________________________________________________________________________     ##STR5##                   %     Relative Ratio                                                                        Sigma.sub.p                         R.sup.B =                  Recovery                                                                            Against #7                                                                            Value                                __________________________________________________________________________       ##STR6##                86    1.34    0.78                                 2                                                                                ##STR7##                82    1.28    0.23                                 3                                                                                ##STR8##                81    1.27    0.57                                 4                                                                                ##STR9##                76    1.19    0.09                                 5                                                                                ##STR10##               72    1.13    0.45                                 6                                                                                ##STR11##               72    1.13    0.00                                 7                                                                                ##STR12##               64    1.00    -0.00                                8                                                                                ##STR13##               69    1.08    -0.07                                9                                                                                ##STR14##               65    1.02    -0.17                                __________________________________________________________________________

The data in the above table show the trend that significantly higherchemiluminescent count is obtainable with 2,6-dimethyl m or psubstituted phenyl ester where the substitution has a σ_(p) valuegreater than 0. The data are also noteworthy in distinguishing nitrosubstitution from other substituents. Though compounds 3 and 8 differmaterially in hydrolytic stability, they each contain large substituentsin the meta or para position. This suggests that bulk alone is not thecontributing factor. In fact, in this regard, nitro, a smallsubstituent, makes the greatest contribution. In the case of compound 3,the sulfonamide linkage within the large substituent provides a highσ_(p) value of 0.4, and in the case of compound 8, the propyl bridge tothe ester contributes to electron donation, hence a σ_(p) value of-0.07. With respect to compound 5, its high σ_(p) value of 0.45 coupledwith the %. Recovery value strongly suggests that the ester hydrolyzedunder the conditions of the experiment.

The chemiluminescent compounds of the present invention have thefollowing schematic formula: ##STR15## In schematic formula 1., thehatched-line box labeled "L" contains the ester, thiolester or amide"linkage" which is carbon-bonded between two substituted rings or ringsystems represented by the circle labeled "Q" and the solid box labeled"R³ ". Whether the linkage L is an ester, thiolester or amide isdetermined by R⁴ being --O--, --S-- or --NT--, respectively. T is astable nitrogen bonded group such as --SO₂ CF₃, to form --N(SO₂ CF₃)--and equivalent groups. The preferred linkage is the ester. M encompassesthe leaving group comprising a portion of L and moiety R³ with itsassociated R¹, R² and R^(2'). M would be the leaving group even if R¹were conjugated to a specific binding material. The leaving grouppossesses the typical pK_(a) of about ≦11.

Q is a heterocyclic ring or ring system to which the ester, thiolesteror amide linkage L is attached at a carbon atom within the heterocyclicring or ring system. That carbon atom (1) is either sp² or sp³hybridized, and (2) is susceptible to attack by peroxide or molecularoxygen to form the intermediate that decays to producechemiluminescence. The oxidation state of the heteroatom within theheterocyclic ring or ring system will determine whether the carbon atomis susceptible to such attack. If the carbon to which the linkage isattached is sp² hybridized, the heteroatom is in a positive oxidationstate (i.e., have a positive charge, for example, as obtained byN-alkylation or N-oxidation). If the carbon to which the linkage isattached is sp³ hybridized, the heteroatom is in a neutral oxidationstate (i.e., uncharged). When the heteroatom is nitrogen, properoxidation states can be achieved only if the nitrogen is substitutedwith an alkyl group (including a reactive functionalized alkyl group),an aryl group (including a reactive functionalized aryl group), --O--(if the nitrogen is in a positive oxidation state) or --OH (if thenitrogen is in a neutral oxidation state). When the heteroatom is inthese "proper" oxidation states, the carbon atom will be susceptible toattack by peroxide or molecular oxygen to produce the chemiluminescentintermediate.

Heterocyclic rings or ring systems that contain the heteroatom in apositive oxidation state include without limitation acridinium,benz[a]acridinium, benz[b]acridinium, benz[c]acridinium, a1,2,4-triazole cation, an isooxazole cation, an isothioazole cation, a1,2-azole cation, an imidazole cation, a benzimidazole cation,quinolinium, isoquinolinium, quinolizinium, a cyclic substitutedquinolinium, pyridinium, pyrimidininium, pyridazinium, pyrazininium,phenanthridinium, and quinoxalinium. Rings or ring systems in which theheteroatom is in a neutral oxidation state include the reduced forms ofthe foregoing. These rings or ring systems are derived from thefollowing rings or ring systems: ##STR16##

The aryl ring or ring system, represented by R³, includes at least onesubstituted six-member ring of the formula ##STR17## in which thesubstituents comprise at least a NO₂ at ring carbons 3, 4 and 5, and R²and R^(2') at ring carbons 2 and 6. The ester, amide or thiolesterlinkage is directly attached through a covalent bond to such six-memberring at ring carbon 1. R³ may include but is not limited to phenyl,naphthyl and anthracyl, which are derivatives of the followingstructures: ##STR18## In those cases where napthyl or anthracyl ringsare employed, one of the rings constitutes R³ and the other ring orrings in combination with it are formed via ring carbons thereof otherthan carbon 1. R³ linked through carbon 1 may be substituted at anyaromatic carbon position provided carbon atoms 2 and 6 are appropriatelysubstituted with electron donating groups and one or more of carbons 3,4 and 5 are appropriately substituted with a group having a σ_(p) valuegreater than 0 and less than 1.

R³ may include, but is not limited to, --NO₂ and the substituent groupsdesignated for R¹, R² and R^(2') described below. However, in thepractice of the invention, the presence of the --NO₂ group is necessary.R³ may be attached through substituents R¹ to protein or other material.In that case, where the attachment is via a nucleophilic substitutionreaction, then the linkage unit attaching R³ and the protein or othermaterial, is typically achieved through the presence of anucleophilically functional R¹ group other than --NO₂.

R² and R^(2') are the classic bulky electron donating groups which arelocated on R³ at C₂ and C₆ so as to sterically hinder, in thetraditional manner, the hydrolysis of the linkage L between R³ and theheterocyclic ring or ring system Q. Where R³ is phenyl with the esterlinkage being attached at position 1, R² and R^(2') are located at theortho 2 and 6 positions. R² and R^(2') may be the same or different, andeither may include:

an alkyl (C₁₋₄) or optionally functionalized

alkyl (C₁₋₄) group an aryl or optionally

functionalized aryl group

--OR, where R is alkyl (C₁₋₄) or aryl

--SR, where R is alkyl (C₁₋₄) or aryl.

The required steric hindrance can also be provided by other rings withina multi-ring R³ which are "adjacent" to the six-member ring to which theester linkage is attached. For example, if R³ is naphthyl and an esterlinkage is attached at the 1 position, R² could be a methyl group at the2 position and R^(2') is the "adjacent" ring containing carbons 7-10. Insuch cases, the adjacent ring is considered, in the classic sense ofsteric hindrance, to be an electron donating substituent (on thesix-member ring within R³) which sterically hinders the hydrolysis ofthe linkage.

R¹ in the preferred embodiment, provides a --NO₂ directly bonded to thearyl carbon atom in one of the meta or para position. It may alsoprovide the capability of entering into a bonding relationship with anactive hydrogen containing group such as amino, amido, carboxyl,hydroxyl, thiol, and the like. The bonding capability of R¹ need only besufficient to link the label compound to the active hydrogen containinggroup. Such bonding may be covalent, ionic, hydrogen and otherassociative bonding that would be acceptable for linking with thecomposition containing the active hydrogen to which bonding is desired.

Particularly desirable R¹ groups, other than the --NO₂ group, are thosethat are directly bonded to the R³ ring C₃,4 and/or 5 atoms through anon-carbon and non-oxygen unit. The preferred R¹ groups containnon-carbon and non-oxygen radicals such as N, S, P, B, Si, and the like,bonded to one or more of the C₃,4 and/or 5 atoms or substituted alkoxyand alkyl. Each of these preferred groups frequently contain bonded tothem one or more of oxo (i.e., =O), oxy (i.e., --O--), halogen, andcarbon bonded organic moieties. When all of the free valences of theradicals are saturated with carbon bonded organic, then the group is anonium, such as quaternary ammonium, sulfonium, phosphonium, and thelike. Desirable groups include the following: ##STR19## in which halogenmay be fluorine, chlorine, bromine and iodine, chlorine being the mostpreferred, X is a functional group reactive with active hydrogen, suchas carboxyl halide, sulfonyl halide, and amino. The essential group withrespect to this invention is --NO₂ and the other groups that can be usedwith it include, as preferable choices, --SO₂ Cl, --Br, and --N(CH₃)₃ ⁺.The SO₂ Cl group provides exceptional bonding to proteins and extremelysensitive assay systems.

The heterocyclic ring or ring system may contain substitutions not shownin schematic formula I. When the heterocyclic ring or ring systemcontains substitution, the substitution may be at any position,including the heteroatom. Such rings and ring systems, whether or notsubstituted, are considered herein to be within the meaning of the term"heterocyclic ring or ring system."

Suitable ring substitutions R⁵, may be functional or non-functional.Functionality can be for the purpose of enhancing the hydrolyticstability of the compound or for providing coupling capabilities viahomolytic or heterolytic reactions or other forms of association thatcouple the label compound to its substrate. Such substitutions includethose for the purposes of producing peri-interactions around the linkageL to enhance its hydrolytic stability, providing functionality to thecompound for coupling to proteins and other materials with complementaryfunctionality, and increasing the compound's solubility andchemiluminescent efficiency. Groups useful for associating the compoundto proteins and other materials so that the chemiluminescent labelcompounds of the invention function in a coupled state with theminclude, but are not limited to, the following functionally substitutedmoieties as ##STR20## or oniums (such as quaternary ammoniums,phosphoniums, sulfoniums, and the like), sugars, polyalkylenepolyaminesand polyalkyleneoxide (e.g., polyoxyethylene, polyoxy-1,2-propylene,polyoxy-1,3-propylene, polyoxy-1,2-butylene, etc.), and the like. Otherchains, groups and functionalities useful for attaching compounds of thepresent invention to protein are discussed in Ji, "BifunctionalReagents," Meth. Enzymology 91:580 (1983), which is incorporated hereinby reference. Methods of joining such attaching groups to protein andother materials utilize both covalent bonding and weaker chemicalforces, and are well known in the art.

These functional groups may be bonded to the heterocyclic ring by acarbon to carbon bond, an oxygen to carbon bond, a nitrogen to carbonbond, and the like. It is desired that the hydrolytic stability of thebond to the heterocyclic ring be greater than that of the linkageachieved through reaction of the functional group and its complementarygroup on the substrate to which the label is being affixed.

Peri substituents, which can cause peri-interactions, include any groupwhich can cause steric hindrance with respect to the carbon to which theester, thiolester or amide linkage is attached and/or with respect tothe carbon within the ester, thiolester of amide linkage. Preferred perisubstituents include short alkyl groups (e.g., methyl, ethyl, and thelike), aryl groups (e.g., phenyl), alkaryl (e.g., tolyl, xylyl, and thelike), alkoxyalkyl (e.g., methoxymethyl, ethoxyethyl, and the like). Theperi substituents, if present, are located on carbon atoms within theheterocyclic ring or ring system which are "adjacent to" the carbon towhich the ester, thiolester or amide L linkage is attached. Moieties caninclude more than one peri substituent. For example, peri substituentscan be placed in the following positions:

(a) in acridiniums and acridans: on C₁ and C₈ ;

(b) in phenanthridiniums and reduced phenanthridiniums: on C₇ ; and

(c) in quinoliniums and reduced quinoliniums: on C₃.

As noted above, covalent or ionic attachment to proteins and othermaterials can be effected through substitutions on R³ or Q's R⁵.

The novel esters, thiolesters and amides of the invention are producedby conventional procedures in the art. For an example, a heterocyclicacyl halide of the formula ##STR21## may be reacted with an aromatic1-hydroxy or 1-mercapto containing the desired diortho (2,6)substitution, to form the desired L linkage. In some cases, the aromatichydroxy or mercapto will contain, as well, the R¹ functionality. Inother cases, it will be necessary to react the resultant esters,thiolesters and amides with reagents suitable for introducing the R¹functionality.

In many cases, the reactions will proceed to the formation ofintermediates that require separation for the next reaction step orfinal products that require isolation. In such cases, conventionaltechniques such as distillation, extraction, crystallization, washingand the like, will be required. Conventional separation by the additionof non-solvent to a solvent solution to force precipitation of a desiredmaterial is frequently found useful.

The above-described improved chemiluminescent compounds are useful in abroad range of specific binding assays for the presence of analyte in asample. "Presence" shall mean herein the qualitative and/or quantitativedetection of an analyte. Such assays may be directed at any analytewhich may be detected by use of the improved chemiluminescent compoundin conjunction with specific binding reactions to form a moiety thereon.These assays include, without limitation, immunoassays, protein bindingassays and nucleic acid hybridization assays.

In a typical immunoassay, the analyte is immunoreactive and its presencein a sample may be determined by virtue of its immunoreaction with anassay reagent. In a typical protein binding assay, the presence ofanalyte in a sample is determined by the specific binding reactivity ofthe analyte with an assay reagent where the reactivity is other thanimmunoreactivity. Examples of this include enzyme-substrate recognitionand the binding affinity of avidin for biotin. In the typical nucleicacid hybridization assay, the presence of analyte in a sample isdetermined by a hybridization reaction of the analyte with an assayreagent. Analyte nucleic acid (usually present as double stranded DNA orRNA) is usually first converted to a single stranded form andimmobilized onto a carrier (e.g., nitrocellulose paper). The analytenucleic acid may alternatively be electrophoresed into a gel matrix. Theimmobilized analyte may then be hybridized (i.e., specifically bound) bya complementary sequence of nucleic acid.

The foregoing specific binding assays may be performed in a wide varietyof assay formats. These assay formats fall within two broad categories.In the first category, the assay utilizes a chemiluminescent conjugatewhich comprises the improved chemiluminescent moiety attached to aspecific binding material. "Specific binding material" means herein anymaterial which will bind specifically by an immunoreaction, proteinbinding reaction, nucleic acid hybridization reaction, and any otherreaction in which the material reacts specifically with a restrictedclass of biological, biochemical or chemical species. In this categoryof assays, the chemiluminescent conjugate participates in a specificbinding reaction and the presence of analyte in the sample isproportional to the formation of one or more specific binding reactionproducts containing the chemiluminescent conjugate. The assay isperformed by allowing the requisite specific binding reactions to occurunder suitable reaction conditions. The formation of specific bindingreaction products containing the chemiluminescent conjugate isdetermined by measuring the chemiluminescence of such productscontaining the chemiluminescent conjugate or by measuring thechemiluminescence of unreacted or partially reacted chemiluminescentconjugate not contained in such products.

This first category of assay formats is illustrated by sandwich assays,competitive assays, surface antigen assays, sequential saturationassays, competitive displacement assays and quenching assays.

In a sandwich format, the specific binding material to which thechemiluminescent moiety is attached, is capable of specifically bindingwith the analyte. The assay further utilizes a reactant which is capableof specifically binding with the analyte to form areactant-analyte-chemiluminescent conjugate complex. The reactant may beattached to a solid phase, including without limitation, dip sticks,beads, tubes, paper or polymer sheets. In such cases, the presence ofanalyte in a sample will be proportional to the chemiluminescence of thesolid phase after the specific binding reactions are completed. Suchassay formats are discussed further in U.S. Pat. Nos. 4,652,533,4,383,031, 4,380,580 and 4,226,993, which are incorporated herein byreference.

In a competitive format, the assay utilizes a reactant which is capableof specifically binding with the analyte to form an analyte-reactantcomplex and with the specific binding material, to which thechemiluminescent moiety is attached, to form a chemiluminescentconjugate-reactant complex. The reactant may be attached to a solidphase, or alternatively reaction products containing the reactant may beprecipitated by use of a second antibody or by other known means. Inthis competitive format, the presence of analyte is "proportional,"i.e., inversely proportional, to the chemiluminescence of the solidphase or precipitate. A further discussion of this assay format may befound in the immediately above mentioned U.S. patents.

In another assay format, the analyte may occur on or be bound to alarger biological, biochemical or chemical species. This type of formatis illustrated by a surface antigen assay. In this format, the specificbinding material is capable of specifically binding with the analyte andthe presence of analyte is proportional to the analyte-chemiluminescentconjugate complex formed as a reaction product. This is illustrated byattaching the chemiluminescent moiety to an antibody which is specificto a surface antigen on a cell. The presence of the cell surface antigenwill be indicated by the chemiluminescence of the cells after thecompletion of the reaction. The cells themselves may be used inconjunction with a filtration system to separate theanalyte-chemiluminescent conjugate complex which is formed on thesurface of the cell from unreacted chemiluminescent conjugate. This isdiscussed further in U.S. Pat. No. 4,652,533.

The improved chemiluminescent moiety may be used in additional assayformats known in the art including without limitation sequentialsaturation and competitive displacement, both of which utilize achemiluminescent conjugate where both (1) the specific binding material,to which the moiety is attached, and (2) the analyte specifically bindwith a reactant. In the case of sequential saturation, the analyte isreacted with the reactant first, followed by a reaction of thechemiluminescent conjugate with remaining unreacted reactant. In thecase of competitive displacement, the chemiluminescent conjugatecompetitively displaces analyte which has already bound to the reactant.

In a quenching format, the assay utilizes a reactant which is capable ofspecifically binding with the analyte to form an analyte-reactantcomplex and with the specific binding material, to which thechemiluminescent moiety is attached, to form a chemiluminescentconjugate-reactant complex. A quenching moiety is attached to thereactant. When brought into close proximity to the chemiluminescentmoiety, the quenching moiety reduces or quenches the chemiluminescenceof the chemiluminescent moiety. In this quenching format, the presenceof analyte is proportional to the chemiluminescence of thechemiluminescent moiety. A further discussion of this format may befound in U.S. Pat. Nos. 4,220,450 and 4,277,437, which are incorporatedherein by reference.

In consideration of the above discussed assay formats, and in theformats to be discussed below, the order in which assay reagents areadded and reacted may vary widely as is well known in the art. Forexample, in a sandwich assay, the reactant bound to a solid phase may bereacted with an analyte contained in a sample and after this reactionthe solid phase containing complexed analyte may be separated from theremaining sample. After this separation step, the chemiluminescentconjugate may be reacted with the complex on the solid phase.Alternatively, the solid phase, sample and chemiluminescent conjugatemay be added together simultaneously and reacted prior to separation. Asa still further but less preferred alternative, the analyte in thesample and the chemiluminescent conjugate may be reacted prior toaddition of the reactant on the solid phase. Similar variations in themixing and reaction steps are possible for competitive assay formats aswell as other formats known in the art. "Allowing under suitableconditions substantial formation" of specific binding reaction productsshall herein include the many different variations on the order ofaddition and reaction of assay reagents.

In the second category of assay formats, the assay utilizes anunconjugated improved chemiluminescent compound. The presence of analytein the sample is proportional to the formation of one or more specificbinding reaction products which do not themselves contain thechemiluminescent moiety. Instead, the chemiluminescent compoundchemiluminesces in proportion to the formation of such reactionproducts.

In one example of this second category of assays, the assay utilizes areactant capable of binding with the analyte to form an analyte-reactantcomplex which causes the chemiluminescent compound to chemiluminesce.This is illustrated by a simple enzyme-substrate assay in which theanalyte is the substrate glucose and the reactant is the enzyme glucoseoxidase. Formation of the enzyme-substrate complex triggers thechemiluminescent compound. Such enzyme-substrate assay for glucose isdisclosed in U.S. Pat. Nos. 3,964,870 and 4,427,770, both of which areincorporated herein by reference. This enzyme-substrate assay is aspecific binding assay in the sense that the substrate specificallybinds to the active site of the enzyme in much the same way that anantigen binds to an antibody. In this assay, the enzyme specificallybinds with the substrate which results in the production of peroxidewhich, in turn, causes the chemiluminescent compound to chemiluminesce.

Also included in the second category of assays are those assays in whichthe formation of the reaction products promotes or inhibitschemiluminescence by the chemiluminescent compound in a less directmanner. In this assay, a first reactant, which is cross reactive withthe analyte, is attached to an enzyme such as glucose oxidase close toits active site. A second reactant which is specific for both theanalyte and the immunoreactive material is added to the sample and thealtered enzyme in the presence of the substrate (i.e., glucose). Whenthe second reactant binds to the first reactant located near the activesite on the enzyme, the second reactant blocks the active site in a waythat the substrate cannot bind to the enzyme at the active site or thebinding of the substrate at the active site is significantly decreased.The second reactant blocking the enzyme in this manner inhibits theenzyme from producing peroxide which, in turn, would have triggered thechemiluminescent moiety. Analyte in the sample, however, will tie up thesecond reactant, thus preventing the second reactant from inhibiting theproduction of peroxide. The presence of analyte will be proportional tothe chemiluminescence of the compound.

The assays contained in the above two categories of assay formats may beheterogeneous or homogeneous. In heterogeneous assays, the reactionproducts, whose formation is proportional to the presence of analyte inthe sample, are separated from other products of the reaction.Separation can be achieved by any means, including without limitation,separation of a liquid phase from a solid phase by filtration,microfiltration, double antibody precipitation, centrifugation, sizeexclusion chromatography, removal of a solid phase (e.g., a dip stick)from a sample solution or electrophoresis. For example, in a sandwichassay the reactant-analyte-chemiluminescent conjugate complex isseparated from unreacted chemiluminescent conjugate. In a surfaceantigen assay, the analyte-chemiluminescent conjugate complex isseparated from unreacted chemiluminescent conjugate. In a competitiveassay, the reactant-chemiluminescent conjugate complex is separated fromunreacted chemiluminescent conjugate. In a sequential saturation assayand in a competitive displacement assay, the reactant-chemiluminescentconjugate complex is separated from unreacted chemiluminescentconjugate. Alternatively, in homogeneous assays the reaction productsare not separated. After the assay reagents have been allowed to react,the chemiluminescence may be measured from the whole assay mixturewhether such mixture is in solution, on a solid phase or distributedbetween various membrane layers of a dip stick or other solid support.The glucose assay using glucose oxidase and a chemiluminescent moietyillustrates a simple homogeneous assay in which separation isunnecessary. The quenching assay illustrates a more complex homogeneousassay in which separation is unnecessary. It is contemplated that eithercategory of assay formats may give rise to either heterogeneous orhomogeneous formats.

Finally, "measuring the chemiluminescence" shall include, whererelevant, the act of separating those specific binding reactionproducts, the formation of which are proportional to the presence ofanalyte in the sample, from other reaction products. It shall alsoinclude, where relevant, the acts of triggering the chemiluminescentmoiety to chemiluminesce in the case of those assay formats in which theformation of the reaction products does not itself trigger thechemiluminescent moiety.

SYNTHESIS OF MOIETIES

The following examples show the synthesis of certain chemiluminescentmoieties of the present invention. These chemiluminescent moieties aretypically made in small quantities and the procedures employed for theirmanufacture do not reflect conventional large scale chemicalmanufacturing procedures. In these reactions, conventional reactionshave been employed to produce the chemiluminescent labels of theinvention. Purification procedures suitable for isolating product areconventional laboratory procedures, such as crystallization out ofsolvent solution by the addition of a nonsolvent, solvent extraction,and the like. In such cases, many different solvents and nonsolvents aresuitable. Yields are the amounts recovered as a percentage of reactantsemployed.

EXAMPLE 1

An illustrative chemiluminescent moiety of the present invention is(2,6-dimethyl-4-nitro)phenyl-3-(3-succinimidyl-oxycarbonyl)propyloxy-N-methyl-acridinium-9-carboxylate fluorosulfonate (or othercounter ions such as chloride, trifluoroacetate, sulfate, etc.) whichhas the following formula: ##STR22## The compound (2,6-dimethyl-4-nitro)phenyl-3-(3-succinimidyl-oxycarbonyl)propyloxy-N-methyl-acridinium-9-carboxylatefluorosulfonate was synthesized according to the following scheme:##STR23##

Condensation of isatin (1) with resorcinol (2) provides the 3-hydroxyacridine-9-carboxylic acid (3). Reaction with benzyl-4-bromo butyrategives the ester (4) with the 3-hydroxy group etherified. Hydrolysisusing base removes both the benzyl groups resulting in the dicarboxylicacid (5). Selective rebenzylation of the carboxylic function of thepropyloxy group gives 9-carboxylic acid (6). Esterification with2,6-dimethyl-4-nitrophenol yields (7) and methylation of the acridinenitrogen gives (8). Deprotection of the carboxyl group with HBr provides(9) and condensation with N-hydroxysuccinimide using DCC provides(2,6-dimethyl-4-nitro)phenyl-3-(3-succinimidyl-oxycarbonyl)propyloxy-N-methyl-acridinium-9-carboxylate fluorosulfonate (10). Thesereactions are described in further detail in the following.

In the first step, 4-bromobutyryl chloride (13.8 g, 75 mmole) was placedin a 100 ml round bottom flask. The flask was cooled to -20° C. using adry ice/carbon tetrachloride bath. Ethyl acetate (50 ml) containingN-methylmorpholine (7.58 g, 75 mmole, 8.2 ml) was added carefully. Usingan addition funnel, benzyl alcohol (6.97 g, 6.67 ml, 6.64 mmoles) wasadded dropwise. After the addition the bath was removed and the mixturewas stirred for 2 hours. The product was transferred to a separatoryfunnel using ethyl acetate (50 ml), washed once with sodium bicarbonate(10%), then twice with water, and dried with anhydrous sodium sulfate.Evaporation of solvents gave benzyl-4-bromo-butyrate as an oil(yield=91%).

Isatin (1) (1.88 g) was slowly added to a solution of potassiumhydroxide (5.07 g, 0.09 mole) dissolved in water (3.5 ml). The reactionflask was heated to about 50° C. in an oil bath. About 10 ml more waterwas added dropwise. Resorcinal (2) (10 g, 0.089 mole) was added and thetemperature was raised to 100° C. as stirring was continued, resultingin the formation of a molten mixture. More isatin (1) (1.88 g) wasadded. The reaction flask (3-necked round bottom) was attached to anitrogen inlet and the water vapors were flushed out by the stream ofnitrogen. The mixture was stirred for 4 hours at 125° C. Water (70 ml)was added and the contents were dissolved by continued stirring. Aftertransferring the mixture to an erlenmeyer flask the volume was broughtup to 200 ml with water. The pH was adjusted to 2.0 with concentratedhydrochloric acid. Filtration and washing of the solids with water gavethe crude acridine acid. It was then dissolved in 2N NaOH (100 ml) andthe solution was filtered through celite. The celite bed was washed with200 ml of 1N NaOH. The filtrate was acidified with concentrated HCl topH 2.0. The precipitate of 2-hydroxy-acridine-9-carboxylic acid (3) wasfiltered, washed with water and was dried in vacuum over P₂ O₅(yield=42%).

In the next sequence, 3-hydroxy-9-acridine carboxylic acid (3) (4 g,0.017 mole), benzyl-4-bromobutyrate (14.6 g, 0.057 mole) and cesiumcarbonate (22.16 g, 0.068 mole) were dissolved in DMSO (125 ml) in a 250ml round bottom flask. The flask was warmed to about 50° C. in an oilbath. After stirring the mixture at that temperature for 1 hour, themixture was poured into water (1 liter). The precipitated product wasextracted with chloroform after making the aqueous suspension basic withsodium bicarbonate. Drying and evaporation of chloroform gave3-(3-benzyloxycarbonyl) -propyloxy-9-(3-benzyloxy-carbonyl-propyl)acridine carboxylate (4) which was chromatographed on a silica gelcolumn using chloroform as solvent. Fractions with R_(f) value of 0.36on TLC with CHCl₃ /EtOAc, 9/1, were pooled. The solvents were evaporated(yield=55%).

Then, 3-(3-benzyloxycarbonyl)-propyloxy-9-(3-benzyloxycarbonyl propyl)acridine carboxylate (4) (4.93 g, 8.3 mmole) was added to a mixture of2N NaOH (300 ml) and methanol (300 ml). The mixture was stirred at roomtemperature for 48 hours. The methanol was removed on a rotaryevaporator and the solution was acidified with concentrated hydrochloricacid to pH 6.0. The precipitated solids were filtered, washed with waterand dissolved in ethyl acetate. The solution was dried and then thesolvents were evaporated to give 3-(3-carboxy)propyloxy-acridine-9-carboxylic acid (5) (yield=92.8%).

This compound, 3-(3-carboxy)propyloxy-acridine-9-carboxylic acid (5)(1.5 g, 4.6 mmole), was dissolved in DMAP (80 ml,1,3-dimethyl-3,4,5,6-tetrahydro-2(IH)pyrimidone) with warming. Benzylalcohol (0.5 ml, 0.52 g, 4.8 mmole), 1,3-dicyclohexylcarbodiimide (1.04g, 5.0 mmole) and N,N-dimethyl aminopyridine (0.2 g, 1.6 mmole) wereadded to the reaction which was previously cooled in a bath of dryice/CCl₄. The mixture was stirred for 15 hours with a nitrogen inlet atroom temperature. The mixture was added to saturated sodium chloride(320 ml). 3-(3-benzyloxycarbonyl) propyloxy -acridine-9-carboxylic acid(6) was filtered and was washed with a small amount of water. Theproduct was chromatographed on a silica gel column using CHCl₃ /MeOH,95/5 as solvent (yield=26%).

The compound 3-(3-benzyloxycarbonyl) propyloxy-acridine-9-carboxylicacid (6) (0.5 g, 1.2 mmole) and p-toluene sulfonyl chloride (0.46 g, 2.4mmole) were dissolved in pyridine (20 ml). The solution was cooled in abath of dry ice/CCl₄ for 15 minutes. 2,6-dimethyl-4-nitrophenol (0.2 gm,1.2 mmole) was added and the cooling bath was removed and the mixturewas stirred for 15 hours at room temperature. It was added to water (450ml) and the pH was adjusted to 2.0 with concentrated hydrochloric acid.The product was filtered, washed with water and was dried in vacuum. Thecrude product was chromatographed on a silica gel column usingchloroform as solvent. Fractions with an R_(f) value of 0.8 on TLC withCHCl₃ /EtOAc, 1:1, were pooled. Evaporation of solvents gave(2,6-dimethyl-4-nitro)phenyl-3-(3-benzyloxycarbonyl)-propyloxy-acridine-9-carboxylate (7)(yield=47%).

The acridine (7) (0.32 g, 0.56 mmole) was dissolved in anhydrousmethylene chloride (4 ml) and methyl fluorosulfate (0.27 ml, 3.36 mmole,6 molar equivalent) was added. The mixture was stirred for 15 hours atroom temperature. Anhydrous ether (20 ml) was added.(2,6-dimethyl-4-nitro)phenyl-3-(3-benzyloxycarbonyl)propyloxy-acridinium-9-carboxylate fluorosulfonate (8) was filtered andwashed with ether (50 ml). The yield was quantitative.

The benzyl-protected acridinium ester (8) (250 ng) was treated with 30%HBr/Acetic acid (3 ml) for 2 hours at 55° C. Anhydrous ether (20 ml) wasadded to precipitate the product. Filtration and washing of the solidswith ether gave (2,6-dimethyl-4-nitro)phenyl-3-(3-carboxyl)propyloxy-acridinium-9-carboxylate fluorosulfonate (9). Crystallizationfrom acetonitrile provided the pure compound (yield=80%). Purificationby crystallization from other solvents achieves equivalent results. Thesame results may be achieved by crystallization from acetonitrile by theaddition of ethyl acetate. Other useful solvent combinations includealcohol and ether, such as methanol, ethanol or propanol and diethylether (ether is the precipitating solvent).

The deprotected acridinium (9) (67 mg, 0.13 mmole) in a 50 ml 2-neckedround bottom flask was dissolved in anhydrous acetonitrile (10 ml).Dicyclohexylcarbodiimide (DCC, 33 mg, 0.16 mmole) was added and themixture stirred for 45 minutes at room temperature. N-hydroxysuccinimide(17 mg, 0.15 mmole) was added and reaction continued for 2.5 hours. MoreDCC (14 mg) and N-hydroxysuccinimide (8 mg) were added and followedagain by the same amounts after 1.5 hours. After 1.5 hours after thelast addition, glacial acetic acid (1.7 ml) was added to quench excessDCC. The solvent was removed in vacuo.

The crude product was purified on a semi-preparative C₁₈ -Dynamax HPLCcolumn (commercially available from Rainin Instrument Co., Inc., Woburn,Mass.) using CH₃ CN/H₂ O (0.1% Trifluoroacetic acid) 60/40, as themobile phase at a flow rate of 1.8 ml/min, using 360 nm for detection.The fraction at retention time 9.4 minutes was collected and thesolvents were removed in vacuo. The(2,6-dimethyl-4-nitro)-phenyl-3-(3-succinimidyl-oxycarbonyl)propyloxy-acridinium-9-carboxylate fluorosulfonate (10) was dried undervacuum in a dessicator containing phosphorus pentoxide (yield=33%). MS:FAB, thioglycerol matrix, 586 (M⁺). HPLC: Rainin C₁₈ Dynamax (10 mm×25mm), CH₃ CN/H₂ O (0.1% trifluoroacetic acid), 60:40, flow rate 1.8ml/min, retention time 9.4 min, detected at 360 nm.

EXAMPLE 2

Further illustrative of the present invention is(2,6-dimethyl-4-nitro)phenyl-3-(3-succinimidyloxycarbonyl)propyloxy-9,10-dihydro-N-methyl-acridan-9-carboxylate which has thefollowing formula: ##STR24## The compound(2,6-dimethyl-4-nitro)phenyl-3-(3-succinimidyloxycarbonyl)propyloxy-9,10-dihydro-N-methyl-acridan-9-carboxylate was synthesizedfrom the acridinium acid (9). Reduction of the acid (9) with sodiumcyanoborohydride gives the acridan which is then converted to the NHSester by the mixed anhydride method. These reactions are described infurther detail in the following.

The acridinium acid (9) (210 mg, 0.37 mmole) was dissolved in a 1:1mixture of acetonitrile and 0.1M phosphate buffer, pH 5.2 (60 ml). Asolution of sodium cyanoborohydride (190 mg) in acetonitrile (5 ml) wasadded dropwise to the acridinium solution. This results in the bleachingof the yellow color of the solution. Stirring was continued for 15minutes. Acetonitrile (100 ml) was added and the solvents were removedin a rotary evaporator. The residue as a suspension in water isextracted with ethylacetate. The organic layer was washed with water anddried. Removal of solvents gave(2,6-dimethyl-4-nitro)phenyl-3-(3-carboxyl)propyloxy-9,10-dihydro-acridan-9-carboxylate(yield=90%).

The acridan acid (125 mg, 0.255 mmole) and N-methylmorpholine (28 l)were dissolved in anhydrous acetonitrile (15 ml). The mixture was cooledin a CCl₄ /dry ice bath under nitrogen. Isobutylchloroformate (35 μl)was added, the mixture was stirred for 3 minutes andN-hydroxysuccinimide (35 mg) dissolved in acetonitrile (2 ml) was added.After stirring at -20° C. for 15 minutes the CCl₄ /dry ice bath wasremoved and the reaction allowed to warm up to room temperature. After 2hours the solvents were evaporated and the residue extracted into ethylacetate. The insoluble N-methylmorpholine hydrochloride salt was removedby filtration. The filtrate was concentrated and hexane (20 ml) wasadded. Cooling results in crystallization of(2,6-dimethyl-4-nitro)phenyl-3-(3-succinimidyloxy-carbonyl)propyloxy-9,10-dihydro-N-methyl-acridan-9-carboxylate. The crystals werefinally filtered and washed with hexane (yield=70%). MS: FAB,dithiothreitol/dithioerythrytol matrix, 588 (M⁺ +1). HPLC: Waters C₁₈Novapak (3.9 mm×15 mm) (commercially available from MilliporeCorporation, Waters Chromatography Division, Milford, Mass.), CH₃ CN/H₂O (0.1% trifluoracetic acid) 60:40, flow rate 1.0 ml/min, retention time6.34 min, detected at 280 nm.

EXAMPLE 3

Another structure encompassed by the invention is (2,6-dimethyl-4-nitro)phenyl-N-methyl-acridinium-9-carboxylate-3-oxo-butyrimidate chloride,hydrochloride which has the following formula: ##STR25## The compound(2,6-dimethyl-4-nitro)phenyl-N-methyl-acridinium-9-carboxylate-3-oxo-butyrimidatechloride, hydrochloride is synthesized according to the followingscheme: ##STR26## Reaction of 3-hydroxy-9-acridine carboxylic acid (16)with 4-bromobutyronitrile gives an ester (17). Hydrolysis of the esterand reesterification with 2,6-dimethyl-4-nitrophenol provides (19).Methylation with methyl fluorosulfate and conversion of the cyano groupto the imidate ester using hydrogen chloride gas and methanol provides(2,6-dimethyl-4-nitro)phenyl-N-methyl-acridinium-9-carboxylate-3-oxo-butyrimidatechloride, hydrochloride (21). These reaction are described in furtherdetail in the following.

In this reaction, 3-hydroxy-9-acridine carboxylic acid (16) (2 g, 8.4mmole), 4-bromobutyronitrile (5.87 ml, 8.74 g, 34 mmole) and cesiumcarbonate (11.08 g, 34 mmole) were dissolved in anhydrous DMSO (50 ml)in a 100 ml round bottom flask. The mixture was warmed to about 50° C.in a water bath with stirring. After 3 hours the mixture was poured intowater (600 ml). The solids were filtered and were dissolved inchloroform. Drying and evaporation of the solvent gave 3-(3-cyano)propoxyl-acridine-9-carboxylic acid-(3-cyano)propyl ester (17). It wasthen dissolved in toluene (50 ml) and cyclohexane (150 ml) was added.The pure product (17) separated, and was then filtered and dried. Thedried product was purified by thin layer chromatography withethylacetate as the mobile phase (R_(f) =0.58) (yield=78.6%).

The cyanopropyl ester (17) (3.73 g. 10 mmoles) was dissolved in amixture of 0.5N NaOH (90 ml) and methanol (90 ml) and stirred in a waterbath at 60° C. using a reflux condenser for 2.5 hours. The methanol wasremoved in a rotary evaporator and the product was extracted with ethylacetate after acidifying the aqueous phase with concentratedhydrochloric acid. Drying and evaporation of the solvent provides3-(3-cyano) propoxyl-acridine-9-carboxylic acid (18) (yield=80%).

The carboxylic acid (18) (4.62 g, 15 mmole) was dissolved in pyridine(130 ml) and the solution was cooled in a CCl₄ /dry ice bath. Paratoluenesulfonyl chloride (5.8 g, 30 mmole) was added and the bath wasremoved. After 15 minutes of stirring at room temperature,2,6-dimethyl-4-nitrophenol (2.8 g, 16.8 mmole) was added. After 18 hoursat room temperature, water (10 ml) was added and the solvents wereremoved in vacuo. The residue was dissolved in chloroform (200 ml) andthe organic layer was washed with saturated sodium bicarbonate (2×100ml), water (2×100 ml), 1N HCl (1×100 ml) and finally with water (2×100ml). Drying and evaporation of the solvent gave(2,6-dimethyl-4-nitro)phenyl-3-(3-cyano) propoxyl-acridine-9-carboxylate(19) which was chromatographed in a silica gel column usingethylacetate/hexane (7:3) as solvent (yield=74.5%).

The ester (19) (1.6 g, 3.52 mmole) was dissolved in dry methylenechloride (50 ml) and under nitrogen methyl fluorosulfate (1.6 ml, 17.6mmole) was added. The solution was stirred at room temperature for 20hours. Anhydrous ether (100 ml) was added and the precipitated(2,6-dimethyl-4-nitro)phenyl-3-(3-cyano)propoxyl-acridinium-9-carboxylate fluorosulfonate (20) was filtered,washed with ether and dried in vacuo (yield=84.7%).

The acridinium ester (20) (4 mg, 7.4×10⁻³ mmole) was dissolved inmethanol (0.5 ml) in a 5 ml 2-necked flask. Anhydrous hydrogen chloridegas was bubbled carefully for 10 minutes. Anhydrous ether (3 ml) wasadded. The precipitated(2,6-dimethyl-4-nitro)phenyl-N-methyl-acridinium-9-carboxylate-3-oxo-butyrimidatechloride, hydrochloride (21) was collected and washed with ether. Thesolid was dried in vacuum and was stored in a dessicator containingphosphorus pentoxide.

EXAMPLE 4

Another chemiluminescent label of the present invention is(2,6-dimethyl-4-nitro)phenyl-N-methyl-phenanthridinium-6-carboxylatefluorosulfonate which has the following formula: ##STR27## The compound(2,6-dimethyl-4-nitro)phenyl-N-methyl-phenanthridinium-6-carboxylatefluorosulfonate is synthesized according to the following scheme:##STR28##

In the reaction, 2-aminobiphenyl (16.9 g, 0.1 mol) is dissolved inanhydrous pyridine (30 ml) and acetic anhydride (10.5 ml, 0.11 mol)added to the solution. The solution is shaken briefly and cooled to roomtemperature and let stand for 15 hours. After the addition of water (50ml) N-acetyl-2-aminobiphenyl (22) is filtered off and recrystallizedfrom aqueous ethanol to give 19.6 g of white needles (yield=93%).

Then N-acetyl-2-aminobiphenyl (22) (19 g, 0.09 mol) is gently refluxedwith freshly distilled phosphoryl chloride (45 ml, 0.49 mol) for 80minutes. The solution is then cooled in ice and the precipitate(6-methylphenanthridine-hydrochloride) filtered off, dissolved in waterand made alkaline with aqueous ammonia. The solution is then extractedwith ether (4×75 ml). The extract is dried over sodium sulfate and theether is removed in vacuo. The resulting yellow oil dissolves in boilingcyclohexane (400 ml) and on cooling forms white needles of6-methylphenanthridine (23) (yield=63%).

The compound 6-(2-hydroxy-1-hydroxymethylethyl)-phenanthridine (24) maybe prepared by treating 6-methylphenanthridine (23) with formaldehydeaccording to the method of Morgan and Walls, J. Chem. Soc. 34:2447(1931), which is incorporated herein by reference. The compound6-(2-hydroxy-1-hydroxymethylethyl)-phenanthridine (24) is formed aswhite needles (yield=57%).

In the next step, a mixture of6-(2-hydroxy-1-hydroxymethyl)-phenanthridine (24) (6 g, 31 mmoles) andfinely powdered selenium dioxide (3.8 g, 34 mmoles) is refluxed in ethylacetate (125 ml) for 10 hours. The deep red solution that form is thenfiltered while hot through celite, before evaporating to dryness. Theresulting solid is digested in warm 1M hydrochloric acid (125 ml),filtered and partially neutralized with sodium bicarbonate. The initialred precipitate is filtered off before completely neutralizing thesolution. The resulting pale yellow solid is filtered off andrecrystallized from acetone/petroleum ether to give 2.7 g of6-formylphenanthridine (yield=42%).

The compound 6-carboxyphenanthridine (25) is prepared by chromic acidoxidation of 6-formylphenanthridine according to the method of Morganand Walls, J. Chem. Soc., supra. The product (25) is a white powder(yield=60%).

In the next step of the procedure, 6-carboxyphenanthridine (25) (662 mg,3 mmoles) is dissolved in anhydrous pyridine (14 ml) and cooled to 0° C.Para-toluene-sulfonyl chloride (1.15 g, 6 mmoles) is added followed by2,6-dimethyl-4-nitro-phenol (501 mg, 3 mmoles) and the mixture isallowed to stand overnight at 4° C. The resulting brown solution isstirred into iced water. The precipitate, filtered off, waschromatographed on a silica gel using chloroform/hexane (1:1) to obtain(2,6-dimethyl-4-nitro)phenylphenanthridine-6-carboxylate (26)(yield=60%).

In a dry two neck 25 ml round bottom flask the ester (26) (369 mg, 1mmole) is suspended in anhydrous methylene chloride (5 ml). Thesuspension is cooled in a dry ice/CCl₄ bath under nitrogen.Chlorosulfonic acid (342 ml, 6 mmole) is added and stirring continued at-20° C. for 30 minutes. The mixture, after warming slowly to roomtemperature, was stirred for an additional 2 hours. Anhydrous ether (20ml) is added and the precipitated solids filtered and washed with ether.Drying gives(2,6-dimethyl-4-nitro)phenyl-N-methyl-phenanthridinium-6-carboxylatefluorosulfonate (27) (yield=90%).

EXAMPLE 5

The compound 3-(2-carboxyethyl)-N-methyl acridinium(2,6-dimethyl-4-nitrophenyl)-9-carboxylate trifluoromethyl sulfonate(N-hydroxy succinimidyl ester), has the formula: ##STR29##

It is made as follows: 4-bromocinnamic acid (Aldrich Chemical Company)(2 g.) was suspended in methanol (10 ml) and platinum oxide (100 mg) wasadded. The solution was stirred for 4 hours in a hydrogen atmosphere,and the solvent was removed to give a colorless oil (1.8 g, 84%) ms(m/e) 244 (M⁺), 211,184,121.

nmr (ppm) 7.34, 7.32, 7.07, 7.04. (2 doublets, 4H) 3.31, (3H, singlet)2.80, 2.57 (multiplets, 4H).

In the reaction, 4-Bromopropionic acid (5 g) was mixed with formanilide(2.25 g), anhydrous K₂ CO₃ and copper bronze (150 mg) in nitrobenzene(17 ml). The mixture was refluxed for 48 hours and the nitrobenzene thenremoved under vacuum. The resulting brown oil was reflexed in a mixtureof acetic acid and conc. HCI 91:1). Extraction into ether, andevaporation of the dried ether solution gave a dark green oil whichsolidified on standing. The solid was washed with petroleum ether togive a white solid (5 g, 67%) ir 1703 (CO) 3403 (NH) ms (m/e) 241 (M⁺),196,182,167.

The previous compound (1.5 g) in carbon disulfide (8 ml) was addedslowly over ten minutes to a refluxing solution of oxalyl chloride (1.07g) in carbon disulfide (6 ml), and refluxing continued for a further onehour. The mixture was evaporated to dryness and carbon disulfide (12 ml)was added. Aluminum chloride (2.88 g) was added in portions, withstirring, and the reaction refluxed for one hour. After removing thesolvent, water was added and the product extracted into ether. A red oilwas obtained which solidified on standing. (1.3 g, 76%). ir 1734,1677cm⁻¹ (CO) nmr (ppm). The spectra clearly showed the presence, in almostequal amounts, of two isomers. There is no need to separate them, asthey both give the same product in the next stage. Peaks at 7.66 to 6.79(aromatic protons, 8H) multiplets 3.02 to 2.89 (two methylene groups,4H).

The previously obtained mixture of isatins (1 g) was dissolved in 10%aqueous KOH and refluxed for 18 hours. A yellow precipitate was obtainedby acidifying the cooled solution with conc. HCl. The precipitate wascollected by filtration and washed with water, methanol and ether (700mg, 76%) ir (nujol) 1711, 1656 (CO) uv (nm) 388, 370, 358, 340 and 256.nmr (ppm) 12 (broad, 1H) 8.20, 2.18, 8.16, 8.14, 8.13 (2H) 8.06, 8.03(2H) 7.90, 7.88, 7.84, 7.83 (1H) 3.01, 3.08, 3.06 (2H) 2.07, 2.68, 2.66(2H)

Analysis: Found (%) C 66.76, H 5.32, N 4.42. C₁₇ H₁₃ NO₄ requires C66.86, H 5.57, N 4.11.

The acridine carboxylic acid prepared in the previous step (20 mg) wassuspended in re-distilled thionyl chloride (2 ml) and reflexed under anargon atmosphere for 4 hours. The solid (ir 1793 and 1731 cm⁻¹ COCl)which resulted after the removal of the thionyl chloride was dissolvedin pyridine (1 ml) and N-hydroxy succinimide (6.92 mg) was added. Thereaction mixture was stirred under an argon atmosphere for 4 hours, andthen cooled in an ice bath. Para-toluene sulfonyl chloride (21.36 mg)was added, and after stirring for 15 minutes, 2,6-dimethyl-4 nitrophenolwas added. The reaction was left for 2.5 hours and the pyridine wasremoved under high vacuum. A few drops of dilute HCl were added and theresulting precipitate was collected by filtration and washed with waterand dried (18 mg, 60%). ir (Nujol) 1812, 1781 and 1740 cm⁻¹ (CO) uv (nm)373, 353 and 262. Addition of base caused the hydrolysis of the phenol,giving peaks at 430, 392, 355 and 257.

The acridine ester was dissolved in dichloromethane (2 ml) and methyltriflate (0.5 ml) added. After stirring over night at room temperature,the volatile materials were removed under vacuum, and the residuedissolved in acetonitrile. A yellow solid was precipitated on theaddition of ether. ir (Nujol) 1812, 1785 and 1745 cm⁻¹ uv (MeOH, nm)369, 354 and 262. Addition of base gave peaks at 430 (p-nitrophenolate)364, 287 and 257 nm. Thus confirming the structure of3-(2-Carboxyethyl)-N-methyl acridinium(2,6-dimethyl-4-nitrophenyl)-9-carboxylate trifluoromethyl sulfonate(N-hydroxy succinimidyl ester).

EXAMPLE 6

The compound 3-(2-carboxyethyl (N-methyl acridinium(2,6-dimethyl-4-nitrophenyl)-9-carboxylate trifluoromethyl sulfonate2,6-dimethyl-4-nitrophenyl ester has the formula: ##STR30##

It is made as follows: 3-(2-carboxyethyl) acridine 9-carboxylic acid (20mg) was dissolved in pyridine (0.5 ml) and the solution cooled in an icebath. Then p-toluene sulfonyl chloride (51 mg) was added and thesolution stirred for 15 minutes. 2,6-Dimethyl-4-nitrophenyl (23 mg) wasadded and the reaction mixture stirred for 2.5 hours at roomtemperature. Ice and dilute HCl were added and the resulting yellowprecipitate filtered off. Washing with water, ethanol and ether gave ayellow solid (26 mg, 65%). The following analysis was obtained:

ir (nujol) Carbonyl peak at 1746 cm⁻¹.

uv (MeOH, HCl) 365, 348, 285 (sh), 262 nm.

ms (m/e) 594 (M⁺ 1), 445, 427, 390.

The above diester (25 mg) was suspended in dichloromethane (2 ml) andfreshly distilled methyl triflate (0.1 ml) added. The solution becameclear dark yellow after 30 minutes. After 18 hours, dry ether was addedto the solution, resulting in a yellow precipitate. This was collectedby filtration (28 mg, 88%). Its analysis showed:

ir (nujol) Strong carbonyl peak at 1752 cm⁻¹.

uv (MeOH) (acid) 368, 353, 263 nm. (base) 430, 364, 287, 257 nm.

Thus confirming the compound 3-(2-Carboxyethyl(N-methyl acridinium(2,6-dimethyl-4-nitrophenyl)-9-carboxylate trifluoromethyl sulfonate2,6-dimethyl-4-nitrophenyl ester.

The above diester, possessing as it does of a moderately hindered activeester, is suitable for labeling for the analytical purposes describedelsewhere in this application. Labeling of the TSH antibody was carriedout as previously described, to give labelled antibody with a specificactivity substantially identical to that using the previously describedesters, resulting in an equivalent immunoassay.

EXAMPLE 7

The compound2,6-dimethyl-4-nitrophenyl-2-(2-[N-(2,6-dimethyl-4-chlorosulfonyl)phenyl]carboxamidoethyl-N-methylacridinium-9-carboxylatefluorosulfonate has the following formula: ##STR31##

This compound is synthesized from the acridine dicarboxylic acid[2-(2-carboxy)ethyl-acridine-9-carboxylic acid] via a series oftransformations. The route for the preparation of2-(2-carboxy)ethyl-acridine-9-carboxylic acid is shown in the followingreaction sequence. ##STR32##

The route for the preparation of2,6-dimethyl-4-nitrophenyl-2-[2-(2,6-dimethyl-4-chlorosulfonyl-phenylcarbamyl)ethyl]-N-methylacridinium-9-carboxylatefluorosulfonate, title compound of this example, is detailed in thefollowing reaction sequence. ##STR33##

The compositions, p-aminophenylpropionic acid, N-[4-(2-carboxyethyl]anthranilic acid, N-phenyl-4-aminophenylpropionic acid andethyl-N-phenyl-4-aminophenylpropionate, were prepared according to thefirst reaction sequence and all were isolated, purified andcharacterized by IR, N.R.M.S. and U.V.

The isatin that was prepared from N-phenyl-ethyl-p-aminophenylpropionatein two steps, as shown in the first reaction sequence of this Example,resulted in two isomers. They were identified by NMR and IR. The data isas follows:

NMR(360 MH₂, FT, CD₃ OD, d PPM):

7.66 ppm to 6.79 ppm (aromatic protons), --CH₂ from 4.13 ppm to 4.04 ppm(as multiplet), --CH₂ from 3.02 ppm to 2.89 ppm (as two multiplets),another --CH₂ (as two triplets) from 2.68 ppm to 2.60 ppm and --CH₃ asmultiplet from 1.23 ppm to 1.18 ppm.

The compound 2-(2-carboxy)ethylacridine-9-carboxylic acid was preparedfrom the isatin isomers, as indicated above, in aqueous potassiumhydroxide solution. The yield was over 76%. Its characterization isobtainable from the following:

NMR (360MH₂ FT, DMSO, d); [8.20 ppm, 8.17 ppm, 8.15 ppm, 8.14 ppm, 8.12ppm] (2H), [8.05 ppm, 8.03 ppm] (d,2H), [7.90 ppm, 7.88 ppm, 7.84 ppm,7.83 ppm] (1H), [3.10 ppm, 3.08 ppm, 3.06 ppm] (t,2H), [2.70 ppm, 2.68ppm, 2.66 ppm] (t,2H) and a broad peak at 12 ppm (-H).

M.S.(FAB): m/e 296 (M⁺ +1) IR(nujol) carbonyl peaks at 1711 cm⁻¹ and1656 cm⁻¹. U.V. (MeOH) 388 nm, 370 nm, 358 nm, 340 nm, 256 nm, in acidat 362 nm, 346 nm and 272 nm.

    ______________________________________                                        Microanalysis for C.sub.17 H.sub.13 NO.sub.4, C.sub.2 H.sub.5 OH                       C %        H %    N %                                                ______________________________________                                        Expected   66.86        5.57   4.11                                           Found      66.76        5.32   4.42                                           ______________________________________                                    

The compound 2-(2-carboxy)ethyl acridine-9-carboxylic acid waschlorinate with thionyl chloride to give the corresponding acid chlorideusing the procedures described in the preceeding examples.

To a solution of the diacid chloride (200 mg., 0.602 mmoles) in pyridine(4 ml.), 2,6-dimethyaniline (73 mg., 0.603 mmoles) was added. Thereaction was stirred at room temperature (23° C.) for 23 hours. Afteraddition of H₂ O/H⁺, compound precipitated out, which was filtered,dried and washed with ether. Compound2-[2-(2,6-dimethyl-phenylcarbamoyl)ethyl]acridine-9-carboxylic acid inover 90% yield, was obtained. Its analysis showed:

M.S.(FAB): m/e 399 (M⁺ +1) IR (nujol): carbonyl peak at 1651 cm⁻¹.

This compound was converted to2,6-dimethyl-4-nitrophenyl-2-[2-(2,6-dimethylphenylcarbamoyl)ethyl]acridine-9-carboxylate##STR34## by Brewster's method with over 70% yield. The compound waspurified by chromatography. The analysis showed:

UV (MeOH) in H⁺ p_(i) 368 nm, 350 nm, 264 nm and in base, at 430 nm, 392nm, 363 nm and 256 nm.

IR (nujol): carbonyl peaks at 1748 cm.⁻¹ and 1659 cm.⁻¹ NMR (500 MH_(z),CDCl₃, 5): aromatic protons from 8.45 ppm to 6.61 ppm, two triplets at[3.39 ppm, 3.37 ppm, 3.36 ppm], (2H), [2.91 ppm, 2.89 ppm, 2.88 ppm],(2H), a singlet at 2.52 ppm (6H) and another singlet at 2.03 ppm (6H).

N-methylation of the compound ##STR35## with methyl fluorosulfonate,resulted,2,6-dimethyl-4-nitrophenyl-2-[2-(2,6-dimethylphenylcarbamoyl)ethyl]-N-methylacridinium-9-carboxylatewith over 80% yield. Its structure was supported by the following data:

M.S. (FAB), 562 m/e (m⁺ +1) U.V.(MeOH) in H⁺, shows peaks at 374 nm, 355(sh), 266 nm, in OH, at 430 nm, 330 nm (sh), 185 nm, 252 nm.

The compound ##STR36## was converted to2,6-dimethyl-4-nitrophenyl-2-[2-(2,6-dimethyl-4-chlorosulfonyl-phenylcarbamoyl)ethyl]-N-methylacridinium-9-carboxylatefluorosulfonate in chlorosulfonic acid and thionylchloride with over 70%yield. Analysis:

M.S. (FAB): 660 m/e (m⁺ +1) U.V.(MeoH) in H⁺, shows peaks at 373 nm, 356nm, 266 nm, in OH, at 425 nm, 288 nm, 255 nm.

EXAMPLE 8

The following procedure for attaching to protein is generally applicableto chemiluminescent labels of the present invention.

Mouse IgG (Sigma, 1 mg) was dissolved in 0.9 ml phosphate buffer (100mM, pH 8.0, 150 mM). If desired, higher pH may be employed, such as a pHas high as 9.5. The solution was then divided into three equal portionsof 0.33 mg/0.3 ml (0.0022 μmoles). About 0.3 mg of a moiety of thepresent invention was dissolved in about 0.4 ml DMF so as to obtain0.022 μmoles of moiety in 15 μl DMF.

0.022 μmoles of the compound of the present invention was mixed with0.0022 μmoles of IgG in a plastic microcentrifuge tube. After 15minutes, an additional 0.022 μmoles of compound was added to themicrocentrifuge tube (compound to protein molar ratio was 20:1). Afteran additional 15 minutes, excess amounts of the compound of the presentinvention were quenched with lysine HCl solution (10 μl in 100 mM p_(i)buffer, pH 8.0) for 15 minutes.

Alternatively, aliquots of 0.0055 μmoles of the compound of the presentinvention was used instead of 0.022 μmoles (compound to protein molarratio was 5:1).

Biorad glass columns (1 cm×50 cm) (commercially available from Biorad,Chemical Division, Richmond, Calif.) were packed with previously swelledand deaerated Sephadex G-50-80 in phosphate buffer (100 mM, pH 6.3, 150mM NaCl, 0.001% TMS) to a bed volume of 45 ml. The reaction solution wasrun through the columns at a flow rate of 0.3-0.4 ml/min. 0.5 mlfractions were collected. Labelled protein fractions were detected bydiluting 20 μl from each fraction to 1 ml and determining thechemiluminescence produced with 30 μl of the diluted solution. Labelledfractions were then pooled.

The pooled conjugate fractions were dialyzed to improve the purity ofimmunoreactive conjugate. The pooled fractions were dialyzed against 500ml pH 6.3 phosphate buffer (100 mM, pH 6.3, 150 mM NaCl, 0.001% TMS)over a period of 24 hours with three buffer changes.

ASSAY PROTOCOLS EXAMPLE 9 1. Components

A) Labelled Ab: Affinity purified rabbit anti-prolactin conjugated to(2,6-dimethyl-4-nitro)phenyl-N-methyl-acridinium-9-carboxylatefluorosulfonate. Storage buffer: 10 mM phosphate buffer, 100 mM NaCl pH6.0, 0.001% Thimerosal, 0.4% BSA.

B) Capture antibody: Rabbit anti-prolactin (6 μg/ml) as a solid phase onNunc® tubes.

C) Solid-phase coated tubes: Dried Nunc® tubes were prepared as follows:

1) 0.3 ml of the capture antibody per tube at 6 μg/ml in PBS buffer(phosphate buffer saline, pH 7.2-7.4, 10 mM phosphate, 100 mM NaCl, 10mM NaN₃) was pipetted into Nunc® tubes.

2) Tubes were incubated for 18-24 hours.

3) Tubes were washed 2 times with the PBS buffer.

4) Tubes were blocked with 2.0% BSA in PBS buffer. Incubate for <4 hoursat room temperature.

5) Tubes were washed 3 times with PBS buffer.

6) Tubes were dried at room temperature.

7) Tubes were stored in plastic freezer bags at 4° C.

D) Standards: Prepared in horse serum 0, 5, 30, 100 and 200 ng/ml/ml

2. Assay Protocol

1) 25 μl of sample or standard was pipetted into the antibody-coatedtubes.

2) 100 μl of labelled antibody was added.

3) Tubes were vortexed gently.

4) Tubes were incubated for 1 hour at room temperature on a rotator.

5) Tubes were washed 3 times with deionized water.

6) Chemiluminescence was counted for 2 seconds [pump 1: 0.1N HNO₃ +0.25%H₂ O₂ ; pump 2: 0.25N NaOH+0.125% CTAC] in a LumaTag™ Analyzer(commercially available from London Diagnostics, Eden Prairie, Minn.).

Labelling Of TSH Antibody With2,6-dimethyl-4-nitrophenyl-2-[2-(2,6-dimethyl-4-chlorosulfonyl-phenylcarbamoyl)ethyl]-N-methylacridinium-9-carboxylatefluorosulfonate

Anti-TSH antibody was labelled with the compound ##STR37## according tothe method of Example 14 that is described previously for ##STR38## Inthe reaction, 250 μg (24.7 μl) of Anti-TSH antibody and 2×12 excessmoles of the compound were added in 15 minute intervals. At the end theaddition, and after purification, the labeled antibody was diluted to6.25 ml. with the diluent buffer solution and used as stock solution.100 ml of labeled antibody (stock solution) was diluted with 0.6 ml. ofantibody diluent.

TSH assays were carried out using the reagents from a LumaTag™ TSH kitand substituting the above labeled TSH antibody reagent, comparableresults were obtained to that obtained using instead a labeled TSH wherethe label is the compound of Example 4.

From the foregoing, it will be obvious to those skilled in the art thatvarious modifications in the above-described compositions and methodscan be made without departing from the spirit and scope of theinvention. Accordingly, the invention may be embodied in other specificforms without departing from the spirit or essential characteristicsthereof. Present embodiments, therefore, are to be considered in allrespects as illustrative and not restrictive, the scope of the inventionbeing indicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

We claim:
 1. A novel chemiluminescent aryl ester, thiolester or amide ofa carboxylic acid substituted heterocyclic ring that is susceptible tochemical attack to dissociate the heterocyclic ring to a transientcompound, wherein the heterocyclic ring is ring carbon-bonded to thecarbonyl of the ester, thiolester or amide moiety and possesses aheteroatom in an oxidation state that allows chemiluminescence bydissociating a compound at the carbon bonded to the carbonyl that decaysto produce chemiluminescence, the aryl is a ring or ring system that isring carbon-bonded to the oxygen, nitrogen or sulfur of the ester orthiolester, as the case may be, and contains at least three substituentsthereon directly bonded thereto acting in concert to sterically andelectronically hinder hydrolysis of the ester or thiolester linkage, oneof which is --NO₂ in a meta or para position.
 2. A hydrolytically stableheterocyclic composition capable of chemiluminescent properties whenlabeled to a specific binding material by chemically-induceddissociation, comprising(a) an aryl ring, (b) a sterically-hinderedester, thiolester or amide linkage moiety with enhanced hydrolyticstability, and (c) a heterocyclic organic ring moiety, in which(1) thecarbonyl carbon of (b) is covalently bonded to a carbon atom (x) of (c)and the remaining free valence of (b) is carbon bonded to an aromaticring carbon atom (y) of (a), (2) (a) contains at least three substituentgroups hindering hydrolysis of (b), two of which are electron donatingand located on the ring carbon atoms adjacent to (y), the remaindercomprise --NO₂ meta or para carbon bonded directly to (y), and (3) (c)contains(i) at least one ring carbon atom adjacent to said ring carbonatom (x), and (ii) a ring member heteroatom in an oxidation state thatprovides such chemiluminescence properties.
 3. A hydrolytically stableconjugate possessing chemiluminescent properties by chemicaldissociation, comprising a chemiluminescent label bonded to a specificbinding material that contains(a) an aryl ring, (b) asterically-hindered ester, thiolester or amide linkage moiety withenhanced hydrolytic stability, and (c) a heterocyclic organic ringmoiety, in which(1) the carbonyl carbon of (b) is covalently bonded to acarbon atom (x) of (c) and the remaining free valence of (b) is carbonbonded to an aromatic ring carbon atom (y) of (a), (2) (a) contains atleast three substituent groups hindering hydrolysis of (b), two of whichare electron donating and located on the ring carbon atoms adjacent to(y), the remainder comprises --NO₂ meta or para carbon bonded directlyto (y), and (3) (c) contains(i) at least one ring carbon atom adjacentto said ring carbon atom (x), and (ii) a ring member heteroatom that isin an oxidation state whereby reaction of molecular oxygen or a peroxidewith said composition forms an intermediate which decays to producechemiluminescence.
 4. The chemiluminescent labeling composition of claim1 conjugated with a specific binding material.
 5. A chemiluminescentassay comprising the conjugate of claim
 4. 6. A chemiluminescent assaykit comprising the conjugate of claim
 5. 7. A hydrolytically stableheterocyclic composition capable of chemiluminescent properties whenlabeled to a specific binding material, by reaction with peroxide ormolecular oxygen, comprising(a) an aryl ring, (b) a sterically-hinderedester, thiolester or amide linkage moiety with enhanced hydrolyticstability, and (c) a heterocyclic organic ring moiety, in which(1) thecarbonyl carbon of (b) is covalently bonded to a carbon atom (x) of (c)and the remaining free valence of (b) is carbon bonded to an aromaticring carbon atom (y) of (a), (2) (a) contains at least three substituentgroups hindering hydrolysis of (b), two of which are electron donatingand located on the ring carbon atoms adjacent to (y), the remaindercontains --NO₂ meta or para carbon bonded directly to (y), and (3) (c)contains (i) at least one ring carbon atom adjacent to said ringcarbonatom (x), and (ii) a ring member heteroatom in an oxidation statethat provides such chemiluminescence properties.
 8. A hydrolyticallystable conjugate possessing chemiluminescent properties by reaction ofmolecular oxygen or a peroxide therewith, comprising a chemiluminescentlabel bonded to a specific binding material that contains(a) an arylring, (b) a sterically-hindered ester, thiolester or amide linkagemoiety with enhanced hydrolytic stability, and (c) a heterocyclicorganic ring moiety, in which(1) the carbonyl carbon of (b) iscovalently bonded to a carbon atom (x) of (c) and the remaining freevalence of (b) is carbon bonded to an aromatic ring carbon atom (y) of(a), 2) (a) contains at least three substituent groups hinderinghydrolysis of (b), two of which are electron donating and located on thering carbon atoms adjacent to (y), the remainder comprise --NO₂ meta orpara to (y), and (3) (c) contains(i) at least one ring carbon atomadjacent to said ring carbon atom (x), and (ii) a ring member heteroatomthat is in an oxidation state whereby reaction of molecular oxygen or aperoxide with said composition forms an intermediate which decays toproduce chemiluminescence.
 9. An assay for the presence of an analyte ina sample comprising contacting an analyte with thechemiluminescent-labeled specific binding material of claim 8, inducingchemiluminescence by decay of an intermediate formable in the presenceof peroxide or molecular oxygen, and measuring chemiluminescencetherefrom to assay the analyte.
 10. A specific binding assay kitcomprising a vial containing a conjugate possessing chemiluminescentproperties by reaction of molecular oxygen or a peroxide therewith andcontaining the chemiluminescent label bonded to a specific bindingmaterial of claim
 8. 11. The chemiluminescent aryl ester, thiolester oramide composition of claim 1 wherein the composition is of the formula:##STR39##
 12. The novel chemiluminescent compound of claim 1 wherein theheterocyclic ring is from the group consisting of acridinium,benz[a]acridinium, benz[b]acridinium, benz[c]acridinium, a1,2,4-triazole cation, an isooxazole cation, an isothioazole cation, a1,2-azole cation, an imidazole cation, a benzimidazole cation,quinolinium, isoquinolinium, quinolizinium, a cyclic substitutedquinolinium, pyridinium, pyrimidininium, pyridazinium, pyrazininium,phenanthridinium, and quinoxalinium.
 13. The novel chemiluminescentcompound claim 3 wherein the heterocyclic ring is from the groupconsisting of acridinium, benz[a]acridinium, benz[b]acridinium,benz[c]acridinium, a 1,2,4-triazole cation, an isooxazole cation, anisothioazole cation, a 1,2-azole cation, an imidazole cation, abenzimidazole cation, quinolinium, isoquinolinium, quinolizinium, acyclic substituted quinolinium, pyridinium, pyrimidininium,pyridazinium, pyrazininium, phenanthridinium, and quinoxalinium.
 14. Thehydrolytically stable heterocyclic composition of claim 3 wherein theheterocyclic ring is form the group consisting of acridinium,benz[a]acridinium, benz[b]acridinium, benz[c]acridinium, a1,2,4-triazole cation, an isooxazole cation, an isothioazole cation, a1,2-azole cation, an imidazole cation, a benzimidazole cation,quinolinium, isoquinolinium, quinolizinium, a cyclic substitutedquinolinium, pyridinium, pyrimidininium, pyridazinium, pyrazininium,phenanthridinium, and quinoxalinium.
 15. The hydrolytically stableheterocyclic composition of claim 8 wherein the heterocyclic ring isfrom the group consisting of acridinium, benz[a]acridinium,benz[b]acridinium, benz[c]acridinium, a 1,2,4-triazole cation, anisooxazole cation, an isothioazole cation, a 1,2-azole cation, animidazole cation, a benzimidazole cation, quinolinium, isoquinolinium,quinolizinium, a cyclic substituted quinolinium, pyridinium,pyrimidininium, pyridazinium, pyrazininium, phenanthridinium, andquinoxalinium.
 16. The novel chemiluminescent compound of claim 1wherein it has the formula: ##STR40## in which L contains the ester,thiolester or amide linkage which is carbon-bonded between twosubstituted rings or ring systems Q and R³ ; R⁴ is --O--, --S-- or--NT--; T is a stable nitrogen bonded group; M is a leaving groupcomprising a portion of L and moiety R³ with its associated R¹, R² andR^(2') bonded thereto; R³ is an aryl group covalently bonded to R⁴ ; R¹is NO₂ directly bonded meta and/or para to the carbon of R³ bonded to R⁴; R² and R^(2') are bulky electron donating groups bonded ortho to thebond of R⁴ to R³ ; M is a leaving group which possesses a pK_(a) ofabout ≦11; Q is a heterocyclic ring or ring system to which the ester,thiolester or amide linkage L is attached at a carbon atom within theheterocyclic ring or ring system, which carbon atom (1) is either sp² orsp³ hybridized, and (2) is susceptible to attack by peroxide ormolecular oxygen to form the intermediate that decays to producechemiluminescence, the oxidation state of the heteroatom within theheterocyclic ring or ring system determining whether the carbon atom issusceptible to such attack and if the carbon to which the linkage isattached is sp² hybridized, the heteroatom is in a positive oxidationstate, and if the carbon to which the linkage is attached is sp³hybridized, the heteroatom is in a neutral oxidation state, and wherethe heteroatom is nitrogen, the nitrogen is substituted with an alkylgroup or a reactive functionalized alkyl group, an aryl group or areactive functionalized aryl group, --O-- where the nitrogen is in apositive oxidation state or --OH where the nitrogen is in a neutraloxidation state, such that the carbon atom is susceptible to attack byperoxide or molecular oxygen to produce a chemiluminescent intermediate.17. The novel chemiluminescent compound of claim 16 wherein theheterocyclic rings or ring systems that contain the heteroatom in apositive oxidation state are from the group consisting of acridinium,benz[a]acridinium, benz[b]acridinium, benz[c]acridinium, a1,2,4-triazole cation, an isooxazole cation, an isothioazole cation, a1,2-azole cation, an imidazole cation, a benzimidazole cation,quinolinium, isoquinolinium, quinolizinium, a cyclic substitutedquinolinium, pyridinium, pyrimidininium, pyridazinium, pyrazininium,phenanthridinium, and quinoxalinium and the heterocyclic rings or ringsystems in which the heteroatom is in a neutral oxidation state are thereduced forms of acridinium, benz[a]acridinium, benz[b]acridinium,benz[c]acridinium, a 1,2,4-triazole cation, an isooxazole cation, anisothioazole cation, a 1,2-azole cation, an imidazole cation, abenzimidazole cation, quinolinium, isoquinolinium, quinolizinium, acyclic substituted quinolinium, pyridinium, pyrimidininium,pyridazinium, pyrazininium, phenanthridinium, and quinoxalinium.
 18. Thenovel chemiluminescent compound of claim 17 wherein R³ includes at leastone substituted six-member ring of the formula ##STR41## in which thesubstituents comprise at least one R¹ at ring carbons 3, 4 and 5, and R²and R^(2') at ring carbons 2 and 6; R⁴ is directly attached through acovalent bond to the six-member ring at ring carbon 1; R³ is one ofphenyl, naphthyl and anthracyl and in those cases where napthyl oranthracyl rings are employed, one of the rings constitutes R³ and theother ring or rings in combination with it are formed via ring carbonsthereof other than carbon 1.